Drug addiction is a complex neurological disorder likely to involve genetic components. For example, studies in mice have shown that elimination of the gene that encodes the receptor for cannabinoids blunts their "addictive response " to opiates and alcohol. Although many genes have been suspected to contribute to addictive behavior, unique human variants in these genes that unequivocally promote susceptibility to drug addiction have not been identified. So far, most attempts to identify such variants have focused on the protein coding segments of the suspect genes. However, it is quite possible that variants that alter the expression of such candidate genes may also render individuals susceptible to addictive disease. Recently, it has become clear that many human genes are regulated by a new epigenetic mechanism, which involves the specific annealing of a small RNA (microRNA) to a target element in the mRNA. In most cases, the annealing of the microRNA leads to a profound decrease in expression of the target mRNA. In our preliminary studies, we have identified a 50 nucleotide element in the 3'UTR of the human cannabinoid receptor mRNA that is likely to be the target of a microRNA. We have observed that insertion of this element into a luciferase reporter significantly reduces its expression. Importantly, a mutation in the target element that is predicted to compromise the binding of microRNAs abrogates the suppressive effect of the target element. Interestingly, this mutation has previously been identified as a rare polymorphism in African American populations. Thus, our overarching hypothesis is that the cannabinoid receptor is regulated by microRNAs and that variants in the cannabinoid receptor element may influence its expression and thus render the individual susceptible to drug addiction. In our first aim, we will confirm and extend our initial observations and identify the microRNA that mediates repression of the cannabinoid receptor. These studies will be aided by our experience in designing and using "Antagomir" reagents. Antagomirs are modified RNAs that can be introduced into cells and can potently down regulate the expression of specific microRNAs. In our second aim, we will examine whether any human variants in the cannabinoid receptor element correlate with susceptibility to addiction. To do this, we have established collaboration with Drs. Kranzler, Covault and Oncken who have access to DNA from control and affected patient populations. [unreadable] [unreadable] [unreadable]